This application claims priority to Japanese Application Serial No. 60787/2000, filed Mar. 6, 2000.
The present invention relates to a microarray, a method for producing the same using pins, and a method for correcting inter-pin spotting amount errors of the same.
In the fields of molecular biology and biochemistry, biopolymers such as nucleic acids and proteins from organisms are identified and/or fractionated in order to search for useful genes or to diagnose diseases. A hybridization reaction is frequently used as a pretreatment for such process, where a target molecule in a sample is hybridized with a nucleic acid or a protein having a known sequence. For this purpose, microarrays, or so-called biochips or DNA chips, are used on which probes such as DNAs, RNAs or proteins with known sequences are immobilized at predetermined positions.
On a microarray, a plurality of regions called features are defined on which different probes are immobilized. The microarray is placed into a reaction container together with sample DNA or the like to allow the fluorescence-labeled sample DNA to hybridize with the probes immobilized on the respective features of the microarray. Thereafter, the microarray is irradiated with excitation light to measure fluorescent intensity of each feature. Based on the measured fluorescent intensities, the binding levels between the respective probes and the sample DNA are obtained and converted into desired information.
Currently, there are roughly two methods for producing microarrays. One is a method in which oligonucleotides are synthesized on a microarray support, and the other is a method in which samples such as cDNAs are spotted on a microarray support. The latter microarray production method generally uses pins for capturing samples from wells of a well plate storing the samples and immobilizing the samples onto the microarray support. In order to improve speed of producing the microarray, a plurality of pins are used simultaneously. Since many microarray experiments require to measure quantitative differences between the spots, great care should be taken to produce uniform pins and to accurately attach pins to a device not to cause any spotting amount error between the plurality of simultaneously-used pins.
However, as the number of the simultaneously-used pins are increased in order to perform faster microarray production, it becomes more difficult to equalize the spotting amounts of the pins by production of uniform pins and by accurate attachment of the pins to the device (i.e., by hardware means).
In view of the above-described problems, the present invention has objectives of providing a microarray which does not have an influence on measurement results even when there is a difference of spotting amount between pins, a method for producing such microarray, and a method for correcting an inter-pin spotting amount error of a microarray.
The present invention realizes the above-mentioned objectives by software means. Specifically, a particular sample is spotted using all of the pins to generate control spots (inter-pin error correction control spots) as a part of samples to be immobilized upon microarray production. Luminescent intensities of the inter-pin error correction control spots are measured using a microarray reader, thereby obtaining spotting amount error information. Correction parameters for the inter-pin errors are obtained for respective pins based on the obtained spotting amount errors to correct luminescent intensities of spots on the same microarray measured with the microarray reader, thereby obtaining corrected values for the measured values of the respective spots.
In order to correct the luminescent intensity of each spot, it is necessary to specify which spot has been spotted with which pin. The present invention comprises a database for storing information as to which sample has been taken from which well (coordinate positions) of a well plate storing samples to be immobilized on respective spots upon microarray production, and a program for tracing which pin is actually used for spotting that sample.
A microarray according to the present invention comprises a support on which a plurality of sample spots are arranged in a two-dimensional array, wherein the plurality of sample spots comprise a group of sample spots of identical samples as control spots having a predetermined positional relationship which are used for correcting spotting amount errors among sample spots belonging to other groups of sample spots having the same relative positional relationships as that of the group of control spots.
The sample spots belonging to the groups of sample spots may not be provided on the microarray in an adjacent manner. When the sample spots are to be immobilized on the microarray at a pitch narrower than pin intervals of the pins of the spotting device, the control spots are provided at skipped positions on the microarray.
According to a method of the invention for producing a microarray having a support on which a plurality of sample spots are arranged in a two-dimensional array, an operation is repeated in which a plurality of samples are simultaneously spotted on the support by using a spotting device provided with a plurality of pins, wherein the method comprise a step of simultaneously spotting the same samples on the support with all of the pins of the spotting device. The identical samples simultaneously spotted using all of the pins of the spotting device act as control spots for measuring inter-pin spotting amount errors caused by differences between individual pins of the spotting device.
According to a method of the invention for correcting an inter-pin spotting amount error of a microarray produced by repeating an operation of simultaneously spotting a plurality of samples on a support by using a spotting device provided with a plurality of pins, the method comprises the steps of: simultaneously spotting the same samples as controls on the support with all of the pins of the spotting device, where a plurality of samples are spotted on the support with the spotting device; measuring spotting amounts of the controls spotted with the respective pins of the spotting device to obtain correction parameters for inter-pin spotting amount errors; and correcting a measured value of each sample spot on the support by using the obtained correction parameters for the inter-pin spotting amount errors.
The spotting amounts of the controls using respective pins of a spotting device may be measured after a hybridization reaction with a sample or before the hybridization. When the spotting amounts of the controls are to be measured after the hybridization reaction, for example, luminescent intensities from fluorescence-labeled samples hybridized to the controls may be measured as the spotting amounts of the controls. On the other hand, when the spotting amounts of the controls are to be measured before the hybridization reaction, for example, a predetermined level of fluorescent substance may be contained in the controls so that luminescent intensities from the fluorescent substances can be measured as the spotting amounts of the controls.
According to a method for correcting an inter-pin spotting amount error of the microarray, information for identifying the pins used for immobilizing the samples to respective sample spot locations may be obtained via positional information of wells on a well plate, which store the samples to be transferred to a microplate with the pins.